Gram quantities of high-purity human alpha-thrombin are being produced for detailed studies on the protein conformation, enzymic specificity, and biologic functions of this central bioregulatory protease in hemostasis and related physiologic processes. In addition to this covalent 2-chain form with high coagulant activity, essentially noncoagulant, noncovalent 3-chain beta- and 4-chain gamma-thrombin forms are being prepared by limited proteolysis, as well as forms made by selective chemical modification with differential enzymic properties. Current studies are being focus on determining essential features of the thrombin active site, which constitute the protein binding and/or biological recognition site(s). These studies include: comparisons of clotting with nonclotting thrombin forms, active site probes, fibrin(ogen) reactions, antithrombin III/heparin reactions,hirudin binding, and crystallography of hirudin and alpha-thrombin. Related collaborative studies include thrombin denaturation/renaturation experiments and characterization of thrombin receptors on platelets and mitogenically responsive cells. Data from our and collaborative studies strongly suggest that alpha-thrombin has a unique protein binding/recognition site(s), which can explain its biological specificity.